As the premier excitatory synaptic transmitter, glutamate has the potential to influence the function of most neuronal circuits in the retina. In addition, glutamate can also act as a potent neurotoxin when present at a high level in the retinal microenvironment. Therefore, extracellular glutamate needs to be maintained at a low level to ensure a high signal-to-noise ratio for glutamatergic neurotransmission and to protect neurons from excitoxic damage and cell death. The long-term objective of the proposed project is to identify mechanisms that operate to maintain glutamate levels in the retina. Extracellular glutamate level is normally kept low by the action of potent uptake systems present in retinal neurons and Muller (glial) cells, and inadequate clearance of glutamate may result in excitotoxic neuronal loss. Although glial glutamate transporters are known to play a major role in glutamate uptake, their specific contribution to glutamate homeostasis has not been directly examined because glial-specific, glutamate uptake inhibitors are not available. Mice with targeted disruption of glial glutamate transporter genes, GLT-1- and GLAST, provide an alternative means to investigate the function of glial glutamate transporters. The present proposal is concerned with neurochemical and immunolocalization studies in GLAST-knockout mice, and tests the hypothesis that GLAST plays a crucial role in regulating retinal glutamate levels, and in metabolic trafficking in the retina. The specific goals of the proposal are to determine whether normal metabolic signaling, and metabolite transfer from Muller cells to photoreceptors is disrupted by loss of GLAST; to examine whether GLAST-deficiency provokes compensatory changes in other glutamate transporters; to determine whether intracellular and extracellular glutamate levels are altered in GLAST-knockout mice; to determine whether NMDA and AMPA glutamate receptor expression is modified in GLAST-null mice; and finally to examine whether loss of GLAST leads to major changes in GABAergic neurons in the retina. Because extracellular glutamate levels have been reported to be elevated in glaucoma and diabetic retinopathy, the proposed studies are crucial for elucidating the cellular mechanisms responsible for elevation in glutamate levels in the retina, and for considering glutamate transporters as potential therapeutic targets.